A four-column combined solid-phase extraction pretreatment method for non-target mass spectrometric analysis

Abstract

The present application relates to the technical field of environmental sample pretreatment, more particularly to a four-column combined solid-phase extraction pretreatment method for non-target mass spectrometry analysis. The method comprises the following steps: S1. collecting a water sample; S2. activating the solid-phase extraction columns: preparing four groups of solid-phase extraction columns, namely HLB, WAX, WCX and PPL solid-phase extraction columns; and activating the four groups of solid-phase extraction columns respectively; S3. sequentially performing sample loading and extraction: using the HLB, WAX, WCX and PPL solid-phase extraction columns to extract the collected water sample sample in sequence; S4. eluting the solid-phase extraction columns; S5. collecting the eluent, nitrogen blowing to 1 mL, and adding ultrapure water to make up to 2 mL; and S6. filtering the eluent after making up to obtain a non-target mass spectrometry sample to be analyzed. The present application can obtain the filtered liquid of intermediate extraction for spectral analysis, and can cover a wide range of chemical substances for enrichment, has good separation effect, and improves the data accuracy of non-target analysis.

Description

Technical Field

[0001] This invention relates to the field of environmental sample pretreatment technology, and more specifically, to a four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis. Background Technology

[0002] Solid-phase extraction (SPE) is a separation and enrichment technique based on the selective adsorption of target compounds by functional groups immobilized on the surface of an adsorbent. It is a widely used sample pretreatment technique. Its principle is to utilize the specific affinity of the adsorbent to adsorb the target compound, followed by elution and further analysis. Currently, SPE is widely used for sample purification and enrichment, and is combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS) for detection and analysis. As a sample pretreatment method, SPE is widely applied in fields such as biology, medicine, chemical engineering, and environmental science. SPE can purify and enrich complex samples through the specific adsorption properties of its materials, thereby lowering the detection limit and reducing matrix effects in complex samples. With the deepening of environmental research, traditional qualitative and quantitative methods for identifying target analytes are no longer sufficient for emerging chemical pollutants and substances lacking standards. In recent years, mass spectrometry has developed rapidly, giving rise to non-targeted techniques for high-throughput screening and identification of unknown substances. Unlike targeted analysis, which requires chemical standards, non-targeted analysis does not require any information or parameters. It only uses high-resolution mass spectrometry to detect samples and analyzes the test data as needed for the research.

[0003] Non-targeted screening aims to obtain as much information as possible about chemical substances from a complex matrix. It has been widely applied in research on surface water, groundwater, and wastewater, primarily targeting organic pollutants such as pesticides, pharmaceuticals, and personal care products. Samples generally require pretreatment processes such as extraction, enrichment, and purification. Solid-phase extraction (SPE) is the main pretreatment method for water samples. Compared to traditional sample pretreatment methods such as liquid-liquid extraction, SPE offers advantages such as simple operation, short processing time, no need for large amounts of organic solvents, high selectivity, and high enrichment.

[0004] A Chinese patent describes a hybrid online solid-phase extraction (SPE) method adapted for enriching complex water samples. This method involves dissolving C18 packing material, polymer packing material, weak cation exchange packing material, and weak anion exchange packing material in a homogenate, followed by ultrasonic stirring to obtain a dispersion. The dispersion is then poured into a homogenization tube, which is connected to a hybrid online SPE column for packing. After packing, the hybrid online SPE column is coupled to an analytical system. After activation, the hybrid online SPE column enriches and extracts various compounds from complex aquatic samples. However, this method, using a direct tandem SPE pretreatment technique, cannot extract the filtrate from the intermediate SPE column, cannot perform spectroscopic analysis, and the packing material in the aforementioned SPE pretreatment technique cannot completely cover contaminants of different polarities. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides a four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis. This method can obtain the intermediate extraction filtrate for spectral analysis, and can enrich and cover chemical substances over a wide range, resulting in good separation effect and improved data quality for non-targeted analysis.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis includes the following steps:

[0008] S1. Collect water samples: Collect water samples and perform filtration on the water samples;

[0009] S2. Activation of solid phase extraction columns: First, prepare four sets of solid phase extraction columns: HLB solid phase extraction column, WAX solid phase extraction column, WCX solid phase extraction column, and PPL solid phase extraction column; second, activate the four sets of solid phase extraction columns respectively.

[0010] S3. Sequential extraction: The collected water sample is extracted sequentially using an HLB solid-phase extraction column, a WAX solid-phase extraction column, a WCX solid-phase extraction column, and a PPL solid-phase extraction column. The water sample is first filtered through an HLB solid-phase extraction column, and the first filtrate is collected. After filtration is complete, the first filtrate is then filtered through a WAX solid-phase extraction column, and the second filtrate is collected. After filtration is complete, the second filtrate is then filtered through a WCX solid-phase extraction column, and the third filtrate is collected. After filtration is complete, the third filtrate is then filtered through a PPL solid-phase extraction column, and the fourth filtrate is collected.

[0011] S4. Elution of solid phase extraction columns: Elution treatment was performed on the four groups of solid phase extraction columns respectively;

[0012] S5. Collect the eluent: Collect the eluent from the four solid-phase extraction columns respectively, blow it with nitrogen to the set volume, and then add ultrapure water to make up the volume.

[0013] S6. Filtration: The eluent after volume adjustment is filtered to obtain the non-targeted mass spectrometry sample to be analyzed.

[0014] Based on the aforementioned technical means, this invention utilizes a combination of multiple solid-phase extraction (SPE) columns to enrich compounds in water samples. Each SPE column employs an adsorbent with different selectivity. The combination of SPE columns with different polarities enables broad-area enrichment and coverage of chemical substances, resulting in good separation, reduced detection limits, and improved data quality for non-targeted analysis. Water samples are sequentially loaded onto each SPE column, while the intermediate filtrate is collected. The intermediate filtrate and subsequent eluent can be used for spectral and water quality analysis. This invention, based on conventional HLB adsorbent SPE, incorporates four SPE columns with different selectivity for pretreatment before non-targeted analysis. The combination of SPE columns with different polarities enables broad-area enrichment and coverage of chemical substances, reduces detection limits, and improves data quality for non-targeted analysis.

[0015] Furthermore, in step S1, the water sample is filtered using a glass fiber filter membrane.

[0016] Furthermore, in step S1, the pore size of the filter membrane is 0.45 μm.

[0017] Further, in step S2, for the HLB solid-phase extraction column, hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene are used as the matrix of the HLB adsorbent; for the WAX ​​solid-phase extraction column, N-vinylpyrrolidone and divinylbenzene are used as the matrix of the WAX ​​adsorbent, with a weak anion exchange layer bonded to -NHCH3 in the structure, forming a WAX packing material with a high molecular weight weak anion exchange, reverse phase, and mixed mode; for the WCX solid-phase extraction column, polystyrene-divinylbenzene polymer is used as the matrix of the WCX adsorbent, with a structure containing weakly acidic carboxylic acid functional groups; for the PPL solid-phase extraction column, modified styrene-divinylbenzene polymer is used as the matrix of the PPL adsorbent. To screen as many substances as possible, this invention employs a combination of multiple solid-phase extraction columns to enrich compounds in water samples, while simultaneously lowering the detection limit. This invention innovatively uses four adsorbents with different selectivities, employing hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene as HLB packing material. This characteristic packing material can broadly enrich acidic, basic, and neutral analytes. First, an HLB solid-phase extraction column is used for overall adsorption; then, N-vinylpyrrolidone and divinylbenzene are used as the matrix of the WAX ​​adsorbent, with a weak anion exchange layer bonded to -NHCH3 in its structure, forming a high-molecular-weight weak anion exchange, reversed-phase, mixed-mode WAX ​​packing material. This characteristic packing material… WAX solid-phase extraction columns exhibit high selectivity for adsorbing strongly acidic compounds; polystyrene-divinylbenzene polymers are used as the matrix for WCX adsorbents, containing weakly acidic carboxylic acid functional groups, with carboxyl groups providing weak cation exchange capacity. WCX solid-phase extraction columns with this characteristic packing material exhibit high selectivity for adsorbing strongly basic compounds; modified styrene-divinylbenzene polymers are used as the matrix for PPL adsorbents, forming a nonpolar reverse-phase system. PPL solid-phase extraction columns with this characteristic packing material exhibit high selectivity for adsorbing polar compounds. The four-column combination achieves coverage of multipolar substances. Simultaneously, this pretreatment process allows for the collection of intermediate permeate and subsequent eluent for spectral and water quality analysis.

[0018] Further, in step S2, for the HLB solid-phase extraction column, methanol solution and ultrapure water are added sequentially for activation treatment; for the WAX ​​solid-phase extraction column, methanol solution containing 5% ammonia, methanol solution and ultrapure water are added sequentially for activation treatment; for the WCX solid-phase extraction column, methanol solution containing 2% formic acid, methanol solution and ultrapure water are added sequentially for activation treatment; and for the PPL solid-phase extraction column, methanol solution and ultrapure water are added sequentially for activation treatment.

[0019] Furthermore, during the activation process, the first solution is added before a liquid surface is formed, and then another solution is added.

[0020] Furthermore, in step S3, before each sample loading, the large-volume sample loading tube is first rinsed with ultrapure water; during the sample loading process, the flow rate of the filtrate is controlled to form a liquid surface above the solid-phase extraction column to ensure that the target extract can be completely adsorbed.

[0021] Further, in step S4, methanol solution is added to the HLB solid-phase extraction column for elution; methanol solution containing 5% ammonia is added to the WAX ​​solid-phase extraction column for elution; methanol solution containing 2% formic acid is added to the WCX solid-phase extraction column for elution; and methanol solution containing 5% ammonia and methanol solution containing 2% formic acid are added first for elution, and then methanol solution is added for a second elution.

[0022] Furthermore, during the elution process, the vacuum pump is turned on to create a certain negative pressure in the solid-phase extraction device, and the gas valve of the solid-phase extraction device is adjusted to allow the eluent to drip drop by drop.

[0023] Further, in step S5, the eluent from each solid-phase extraction column is collected, then blown to the set volume using a dry nitrogen blower, and then diluted with ultrapure water and shaken evenly.

[0024] Further, in step S6, a needle filter with a 0.22 μm organic phase filter membrane is used for filtration.

[0025] Compared with existing technologies, the beneficial effects are as follows: The present invention provides a four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis, which sets up four solid-phase extraction columns with different selectivity for pretreatment before non-targeted analysis, combines solid-phase extraction columns with different polarities to enrich and cover chemical substances over a wide range, reduce the detection limit, improve the data quality of non-targeted analysis, and can also extract water samples before and after loading solid-phase extraction columns for spectral analysis. Attached Figure Description

[0026] Figure 1 This is a flowchart of the method of the present invention.

[0027] Figure 2 This is a schematic diagram of the method flow of an embodiment of the present invention.

[0028] Figure 3 This is a schematic diagram showing the change in DOC (dissolved organic carbon) concentration and removal rate of water samples after loading in an embodiment of the present invention.

[0029] Figure 4 This is a schematic diagram showing the change in the ultraviolet-visible absorption spectrum of the water sample after loading in an embodiment of the present invention.

[0030] Figure 5This is a schematic diagram of the three-dimensional fluorescence spectrum changes of water samples after loading in an embodiment of the present invention, wherein tyrosine and tyrosine-like substances (region I), tryptophan and tryptophan-like substances (region II), fulvic acid and fulvic acid-like substances (region III), soluble microbial products (region IV), and humic acid and humic acid-like substances (region V). Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The present invention will be described in one embodiment below with reference to specific embodiments. The accompanying drawings are for illustrative purposes only and represent schematic diagrams, not actual pictures, and should not be construed as limiting the present patent. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged, or reduced, and do not represent the actual product size. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0032] In the description of this invention, it should be understood that if terms such as "upper," "lower," "left," and "right" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, they are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms describing positional relationships in the accompanying drawings are only for illustrative purposes and should not be construed as limiting this patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances. In addition, if the embodiments of this invention involve descriptions of "first," "second," etc., such descriptions are only for descriptive purposes and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of those features. Furthermore, the meaning of "and / or" throughout the text is to include three parallel solutions. Taking "A and / or B" as an example, it includes solution A, or solution B, or a solution that simultaneously satisfies A and B.

[0033] Example

[0034] like Figure 1 and Figure 2 As shown, a four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis includes the following steps:

[0035] Step S1. Collect water samples: Collect water samples and filter them.

[0036] Take 1.5L of natural water samples and 1L of high-concentration wastewater samples, place them in pre-cleaned polypropylene bottles, and transport them within 48 hours with ice packs. Store them in a laboratory refrigerator at approximately 4°C. After restoring to room temperature in advance, make up the volume of the water sample using a suitable graduated cylinder. For samples with low concentrations, such as natural water, make up to 1L; for samples with high concentrations, such as wastewater, make up to 0.5L. Filter the water sample using a 0.45μm glass fiber filter membrane after making up the volume. Transfer the filtered water sample to a 1L blue-capped bottle.

[0037] Step S2. Activation of Solid Phase Extraction Columns: First, prepare four sets of solid phase extraction columns: HLB solid phase extraction column, WAX solid phase extraction column, WCX solid phase extraction column, and PPL solid phase extraction column; second, activate each of the four sets of solid phase extraction columns. For the HLB solid phase extraction column, hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene are used as the matrix of the HLB adsorbent; for the WAX ​​solid phase extraction column, N-vinylpyrrolidone and divinylbenzene are used as the matrix of the WAX ​​adsorbent, with a weak anion exchange layer bonded to -NHCH3 in the structure, forming a WAX packing material with high molecular weight weak anion exchange, reverse phase, and mixed mode; for the WCX solid phase extraction column, polystyrene-divinylbenzene polymer is used as the matrix of the WCX adsorbent, with a structure containing weakly acidic carboxylic acid functional groups; for the PPL solid phase extraction column, modified styrene-divinylbenzene polymer is used as the matrix of the PPL adsorbent.

[0038] For a 500 mg / 6 mL HLB solid-phase extraction column, add 6 mL of methanol and 20 mL of ultrapure water sequentially; for a 225 mg WAX solid-phase extraction column, add 6 mL of methanol solution containing 0.5% ammonia (hereinafter referred to as ammonia-ethanol), 6 mL of methanol, and 20 mL of ultrapure water sequentially; for a 225 mg WCX solid-phase extraction column, add 6 mL of methanol solution containing 2% formic acid (hereinafter referred to as formic acid-ethanol), 6 mL of methanol, and 20 mL of ultrapure water sequentially; for a 500 mg / 6 mL PPL solid-phase extraction column, add 6 mL of methanol and 20 mL of ultrapure water sequentially. Filter one liquid after the other is added. The WAX ​​and WCX solid-phase extraction columns do not have column tubes; instead, attach a 5 mL syringe to the top for activation.

[0039] Step S3. Perform sample loading and extraction in sequence: Extract the collected water sample sequentially using an HLB solid-phase extraction column, a WAX solid-phase extraction column, a WCX solid-phase extraction column, and a PPL solid-phase extraction column. First, filter the water sample through an HLB solid-phase extraction column and collect the first filtrate. After complete filtration, filter the first filtrate through a WAX solid-phase extraction column and collect the second filtrate. After complete filtration, filter the second filtrate through a WCX solid-phase extraction column and collect the third filtrate. After complete filtration, filter the third filtrate through a PPL solid-phase extraction column and collect the fourth filtrate. Before each sample loading, the large-volume sample tube was rinsed with ultrapure water. During the sample loading process, the flow rate of the filtrate was controlled at 3 mL / s, and for high-concentration wastewater, the flow rate was controlled at 1 mL / s to form a liquid surface above the solid-phase extraction column, so as to ensure that the target extract can be completely adsorbed and avoid penetrating the adsorbent packing.

[0040] Step S4. Elution of Solid Phase Extraction Columns: Elute the four sets of solid phase extraction columns separately. To elute the solid phase extraction columns, turn on the vacuum pump to create negative pressure, adjusting the pressure to allow the liquid to drip slowly, drop by drop. For the HLB solid phase extraction column, add 6 mL of methanol; for the WAX ​​solid phase extraction column, add 6 mL of ammonia; for the WCX solid phase extraction column, add 6 mL of formic acid alcohol; for the PPL solid phase extraction column, elute two tubes. In the first tube, add 3 mL of ammonia and 3 mL of formic acid alcohol sequentially, filtering out one liquid before adding the other. In the second tube, add 6 mL of methanol.

[0041] Step S5. Collect the eluent: Collect the eluent from the four solid-phase extraction columns respectively, blow it with nitrogen to the set volume, and then add ultrapure water to make up to the volume; blow it with a dry nitrogen blower to 1 mL, and finally make up to 2 mL with ultrapure water, and shake it up and down 10 times to mix.

[0042] Step S6. Filtration: The eluent after volume adjustment is filtered to obtain the non-targeted mass spectrometry analyte sample. The sample is then filtered through a needle filter with a 0.22 μm organic phase filter membrane and bottled.

[0043] In addition, the instruments and consumables used in the experiment of this embodiment include glass fiber filter membrane (pore size 0.45μm), needle filter of organic phase filter membrane (pore size 0.22μm), HLB solid phase extraction column (500mg / 6mL), WAX solid phase extraction column (225mg), WCX solid phase extraction column (225mg), PPL solid phase extraction column (500mg / 6mL), solid phase extraction device (12 wells), and dry nitrogen blowing device (24 positions).

[0044] The methanol, ammonia, and formic acid used in this embodiment were all chromatographically pure (purity ≥ 99.0%), and the ultrapure water used was Milli-Q ultrapure water (18.2 MΩ·cm).

[0045] This invention utilizes a combination of multiple solid-phase extraction (SPE) columns to enrich compounds in water samples. Each SPE column employs an adsorbent with different selectivity. The combination of SPE columns with different polarities enables broad-area enrichment and coverage of chemical substances, resulting in good separation, lower detection limits, and improved data quality for non-targeted analysis. Water samples are sequentially loaded onto each SPE column, while the intermediate filtrate is collected. This intermediate filtrate and subsequent eluent can be used for spectral and water quality analysis. Building upon conventional HLB adsorbent-based SPE, this invention incorporates four SPE columns with different selectivities for pretreatment before non-targeted analysis. The combination of SPE columns with different polarities enables broad-area enrichment and coverage of chemical substances, lowers detection limits, and improves data quality for non-targeted mass spectrometry analysis.

[0046] like Figures 3 to 5 As shown, by Figure 3 It can be seen that the DOC (dissolved organic carbon) of the water sample after filtration was 1.805 mg / L. After each solid-phase extraction column, the DOC of the water sample decreased. After loading the HLB solid-phase extraction column, the DOC of the water sample was 1.253 mg / L, which was about 30% lower than the DOC of the filtered raw water. The DOC of the water sample after loading the four columns was 0.526 mg / L. The coverage of DOC in the water sample by the four columns reached 70.86%, indicating that solid-phase extraction columns of different polarities achieved effective separation and stratified adsorption of dissolved organic carbon with different properties. Figure 4 The specific enrichment of the four-column combined solid-phase extraction (SPE) method was demonstrated from the perspective of UV-Vis absorption spectroscopy. In principle, UV-Vis absorption spectroscopy utilizes the UV-Vis spectrum and absorption degree generated by the absorption of UV and visible light by molecules or ions of a substance to analyze, determine, and infer the composition, content, and structure of the substance. For the same analyte solution, the higher the concentration, the higher the absorbance value. After loading water samples onto HLB, WAX, WCX, and PPL SPE columns, the absorbance values ​​all decreased. The UV254 value is the absorbance of some organic matter in water under 254nm wavelength UV light, reflecting the content of humic macromolecular organic matter and aromatic compounds containing C=C and C=O double bonds. The UV254 value also gradually decreased with the tandem adsorption of the SPE columns, indicating that the four-column combined SPE pretreatment method can effectively adsorb substances of different polarities based on the characteristics of different SPE columns adsorbing substances with different properties, reducing the concentration of organic matter in the water sample, reducing matrix effects, and improving the quality of subsequent non-targeted mass spectrometry analysis. Figure 5This is a three-dimensional fluorescence spectrum of the water sample after loading. The three-dimensional fluorescence spectrum is a matrix spectrum characterized by three-dimensional coordinates of excitation wavelength (y-axis), emission wavelength (x-axis), and fluorescence intensity (z-axis). Different types of soluble fluorescent organic matter are located in different positions, and the fluorescence intensity represents the concentration of soluble fluorescent organic matter. The fluorescence region integration method is commonly used to analyze the fluorescence spectrum. The fluorescence region integration method divides the three-dimensional fluorescence spectrum into different regions, each representing a part of the soluble organic matter, including tyrosine and tyrosine-like substances (region I), tryptophan and tryptophan-like substances (region II), fulvic acid and fulvic acid-like substances (region III), soluble microbial products (region IV), and humic acid and humic acid-like substances (region V). After adsorption over a wide range using the HLB solid-phase extraction column, the overall area of ​​the fluorescence peak region in phase b decreased compared to phase a. After adsorption using the WAX ​​solid-phase extraction column, the areas of the fluorescence peak regions in regions V (humic acid fluorescence peak region) and III (fulvic acid and fulvic acid-like fluorescence peak region) were significantly reduced. After adsorption using the WCX solid-phase extraction column, the area of ​​the protein fluorescence peak region (including regions I, II, and IV) was significantly reduced. Finally, after adsorption using the PPL solid-phase extraction column, the overall fluorescence peak intensity was further reduced. This demonstrates the specific adsorption capability of the four-column combination, reduces the difficulty of subsequent mass spectrometry data analysis, allows for more targeted development of specific methods, and effectively improves the quality of non-targeted mass spectrometry analysis.

[0047] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0048] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis, characterized in that, Includes the following steps: S1. Collect water samples: Collect water samples and perform filtration on the water samples; S2. Activation of solid phase extraction columns: First, prepare four sets of solid phase extraction columns: HLB solid phase extraction column, WAX solid phase extraction column, WCX solid phase extraction column, and PPL solid phase extraction column; second, activate the four sets of solid phase extraction columns respectively. S3. Sequential extraction: The collected water sample is extracted sequentially using an HLB solid-phase extraction column, a WAX solid-phase extraction column, a WCX solid-phase extraction column, and a PPL solid-phase extraction column. The water sample is first filtered through an HLB solid-phase extraction column, and the first filtrate is collected. After filtration is complete, the first filtrate is then filtered through a WAX solid-phase extraction column, and the second filtrate is collected. After filtration is complete, the second filtrate is then filtered through a WCX solid-phase extraction column, and the third filtrate is collected. After filtration is complete, the third filtrate is then filtered through a PPL solid-phase extraction column, and the fourth filtrate is collected. S4. Elution of solid phase extraction columns: Elution treatment was performed on the four groups of solid phase extraction columns respectively; S5. Collect the eluent: Collect the eluent from the four solid-phase extraction columns respectively, blow it with nitrogen to the set volume, and then add ultrapure water to make up the volume. S6. Filtration: The eluent after volume adjustment is filtered to obtain the non-targeted mass spectrometry sample to be analyzed.

2. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 1, characterized in that, In step S1, the water sample is filtered using a glass fiber filter membrane with a pore size of 0.45 μm.

3. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 1, characterized in that, For HLB solid-phase extraction columns, hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene are used as the matrix of HLB adsorbents; for WAX solid-phase extraction columns, N-vinylpyrrolidone and divinylbenzene are used as the matrix of WAX adsorbents, with a weak anion exchange layer bonded to -NHCH3 in the structure, forming a WAX packing material with high molecular weight weak anion exchange, reverse phase, and mixed mode; for WCX solid-phase extraction columns, polystyrene-divinylbenzene polymer is used as the matrix of WCX adsorbents, with a structure containing weakly acidic carboxylic acid functional groups; for PPL solid-phase extraction columns, modified styrene-divinylbenzene polymer is used as the matrix of PPL adsorbents.

4. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 3, characterized in that, In step S2, for the HLB solid-phase extraction column, methanol solution and ultrapure water are added sequentially for activation treatment; for the WAX ​​solid-phase extraction column, methanol solution containing 5% ammonia, methanol solution, and ultrapure water are added sequentially for activation treatment; for the WCX solid-phase extraction column, methanol solution containing 2% formic acid, methanol solution, and ultrapure water are added sequentially for activation treatment; and for the PPL solid-phase extraction column, methanol solution and ultrapure water are added sequentially for activation treatment.

5. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 4, characterized in that, During the activation process, the first solution is added before a liquid surface is formed, and then another solution is added.

6. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 1, characterized in that, In step S3, before each sample loading, the large-volume sample loading tube is first rinsed with ultrapure water; during the sample loading process, the flow rate of the filtrate is controlled to form a liquid surface above the solid-phase extraction column to ensure that the target extract can be completely adsorbed.

7. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 1, characterized in that, In step S4, methanol solution is added to the HLB solid-phase extraction column for elution; methanol solution containing 5% ammonia is added to the WAX ​​solid-phase extraction column for elution; methanol solution containing 2% formic acid is added to the WCX solid-phase extraction column for elution; and methanol solution containing 5% ammonia and 2% formic acid are added first for elution, followed by a second elution with methanol solution.

8. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 7, characterized in that, During the elution process, the vacuum pump is turned on to create a certain negative pressure in the solid phase extraction device, and the gas valve of the solid phase extraction device is adjusted to allow the eluent to drip drop by drop.

9. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 1, characterized in that, In step S5, the eluent from each solid-phase extraction column is collected, then blown to the set volume using a dry nitrogen blower, and then diluted with ultrapure water and shaken up and down to mix.

10. The four-column combined solid-phase extraction pretreatment method for non-targeted mass spectrometry analysis according to claim 1, characterized in that, In step S6, a needle filter with a 0.22 μm organic phase filter membrane is used for filtration.

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